publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Article Mycobacterium tuberculosis Sulfolipid-1 Activates Nociceptive Neurons and Induces Cough Graphical Abstract Highlights d An Mtb organic extract activates nociceptive neurons and induces cough in guinea pigs d Mtb sulfolipid-1 is necessary and sufficient to trigger neuronal activation and cough d Guinea pigs infected with an SL-1-deficient Mtb mutant do not coughMycobacterium tuberculosis produces a glycolipid called sulfolipid-1 (SL-1) that triggers cough by activating nociceptive neurons.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that Mtb can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, Mtb is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secrete virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb secreted proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 secreted proteins from Mtb for their ability to associate with eukaryotic membranes using a unique temperature-sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. We identified five Mtb secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms. IMPORTANCE Advances have been made to identify secreted proteins of Mycobacterium tuberculosis during animal infections. These data, combined with transposon screens identifying genes important for M. tuberculosis virulence, have generated a vast resource of potential M. tuberculosis virulence proteins. However, the function of many of these proteins in M. tuberculosis pathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200 M. tuberculosis secreted proteins for eukaryotic membrane binding, host subcellular localization, and interactions with host vesicular trafficking. In addition, we observed the localization of one secreted protein, Mpt64, to the endoplasmic reticulum (ER) during M. tuberculosis infection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the type VII secretion system. Finally, we observed that Mpt64 impairs the ER-mediated unfolded protein response in macrophages.

BackgroundA hallmark symptom of active pulmonary tuberculosis vital for disease transmission is cough. The current paradigm for tuberculosis-related cough is that it results from airway damage or irritation. However, there is limited experimental data to support this theory, and whether Mycobacterium tuberculosis (Mtb) induces cough to facilitate its own transmission has not been explored. The cough reflex is a complex and coordinated event involving both the nervous and musculoskeletal systems initiated by particulate or chemical molecules activating nociceptive neurons, which sense pain or irritation. This activation induces a signaling cascade ultimately resulting in a cough. Respiratory nociceptive neurons innervate the airway of humans and most mammals, and thus are poised to respond to noxious molecules to help protect the lung from damage. Because Mtb is a lung pathogen, cough is a primary mechanism of Mtb transmission, and respiratory nociceptive neurons activate cough, we hypothesized that Mtb produces molecules that stimulate cough, thereby facilitating its spread from infected to uninfected individuals.MethodsWe used an in vitro neuronal activation bioassay to fractionate, identify, and characterize Mtb cough-inducing molecules. We also measured cough in vivo in response to pure Mtb-derived cough molecules and during Mtb infection using a guinea pig model.ResultsWe found that an acellular organic extract of Mtb triggers and activates nociceptive neurons in vitro with a neuronal response that is as robust as the response to capsaicin, an established nociceptive and cough-inducing molecule. Using analytical chemistry and our neuronal bioassay, we then isolated 2 molecules produced by Mtb that activate nociceptive neurons. Both the organic Mtb extract and purified molecules alone were sufficient to induce cough in a conscious guinea pig cough model. Finally guinea pigs infected with wild-type Mtb cough much more frequently than guinea pigs infected with Mtb strains unable to produce nociceptive molecules.ConclusionWe conclude that Mtb produces molecules that activate nociceptive neurons and induce cough. These findings have significant implications for our understanding of Mtb transmission.Disclosures All authors: No reported disclosures.

word count: 203 22 23 2 Text word count: 7,901 24 3 Abstract 25Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the 26 most successful human pathogens. One reason for its success is that Mtb can reside 27 within host macrophages, a cell type that normally functions to phagocytose and destroy 28 infectious bacteria. However, Mtb is able to evade macrophage defenses in order to 29 survive for prolonged periods of time. Many intracellular pathogens secret virulence 30 factors targeting host membranes and organelles to remodel their intracellular 31 environmental niche. We hypothesized that Mtb exported proteins that target host 32 membranes are vital for Mtb to adapt to and manipulate the host environment for 33 survival. Thus, we characterized 200 exported proteins from Mtb for their ability to 34 associate with eukaryotic membranes using a unique temperature sensitive yeast 35 screen and to manipulate host trafficking pathways using a modified inducible secretion 36 screen. We identified five Mtb exported proteins that both associated with eukaryotic 37 membranes and altered the host secretory pathway. One of these secreted proteins, 38Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human 39 macrophages and was necessary for Mtb survival in primary human macrophages. 40